Phosphatidyl choline (PC), also named lecithin, is one of the major sources of polyunsaturated fatty acids such as arachidonic and linoleic acids. The former is a precursor of eicosanoids which have numerous biological activities. Hydrolysis of PC yields lysophosphatidyl choline (LysoPC) and constituent fatty acids, which have been implicated in signal transduction (Asaoka et al., Proc. Natl. Acad. Sci. USA 90:4917-4921 (1993); Yoshida et al., Proc. Natl. Acad. Sci. USA 89:6443-6446 (1992)). An increasing body of evidence indicates that LysoPC, which is present in high concentrations in oxidized low density lipoproteins may play a significant role in atherogenesis and other inflammatory disorders (Steinberg et al., New. Eng. J. Med. 320:915-924 (1989)). LysoPC has been reported to increase the transcription of genes encoding platelet derived growth factor A and B chains, and heparin-binding epidermal growth factor-like protein (HB-EGF) in cultured endothelial cells (Kume and Gimbrone, J. Clin. Invest. 93:907-911 (1994)), and to increase mRNA encoding HB-EGF in human monocytes (Nakano et al., Proc. Natl. Acad. Sci. USA 91:1069-1073 (1994)). These gene products are mitogens for smooth muscle cells and fibroblasts (Higashiyama et al., Science 251:936-939 (1991); Ross, Nature (Lond.) 362:801-809 (1993)). LysoPC has also been shown to activate protein kinase C in vitro (Sasaki et al., FEBS Letter 320:47-51 (1993)), to potentiate the activation of human T lymphocytes (Asaoka et al., Proc. Natl. Acad. Sci. USA 89:6447-6451 (1992)) and to potentiate the differentiation of HL-60 cells to macrophages induced by either membranepermeable diacylglycerols or phorbol esters (Asaoka et al., Proc. Natl. Acad. Sci. USA 90:4917-4921 (1993)).
LysoPC may also provide a source of bioactive lysophosphatidic acid (1-acyl-sn-glycero-3-phosphate, LysoPA) (Moolenaar et al., Rev. Physiol. Biochem. Pharmacol. 119:47-65 (1992)) through hydrolysis by lysophospholipase D (Tokumara et al., Biochim. Biophys. Acta 875:31-38 (1986)). LysoPA is a naturally occurring phospholipid with a wide range of growth factor-like biological activities. It is well established that LysoPA can act as a precursor of phospholipid biosynthesis in both eukaryotic and prokaryotic cells (Van den Bosch, Ann. Rev. Biochem. 43:243-277 (1974); Racenis et al., J. Bacteriol. 174:5702-5710 (1992)). The ability of LysoPA to act as an intercellular lipid mediator has been noted (Vogt, Arch. Pathol. Pharmakol. 240:124-139 (1960); Xu et al., J. Cell. Phvsiol. 163:441-450 (1995); Xu et al., Biochemistry 309:933-940 (1995); Tigyi et al., Cell Biol. 91:1908-1912 (1994); Panetti et al., J. Lab. Clin. Med. 129(2):208-216 (1997)). LysoPA is rapidly generated by activated platelets and can stimulate platelet aggregation and wound repair.
Ovarian cancer activating factor (OCAF), has been isolated from ovarian cancer ascites fluid (Mills et al., Cancer Res. 48:1066 (1988); Mills et al. J. Clin. Invest. 86:851 (1990) and U.S. Pat. Nos. 5,326,690 and 5,277,917) and has been identified to consist of multiple forms of LysoPA (Xu et al., Clin. Cancer Res. 1:1223-1232 (1995)). LysoPA has been identified as a potent tumor growth factor in the ascites fluid of ovarian cancer patients (Id.)
Other lysophospholipids associated with various conditions include lysophosphatidyl serine (LysoPS), lysophosphatidyl ethanolamine (LysoPE), lysophosphatidyl glycerol (LysoPG and lysophosphatidyl inositol (LysoPI). Activated platelets secrete two kinds of phospholipase: sPLA2 and PS-PLA1. sPLA2 is reported to be elevated in inflammatory reactions and inhibition of this enzyme reduced inflammation (Schrier et al., Arthritis Rheum. 39(8):1292-1299 (1996); Tramposch et al., Pharmacol. and Experimental Therapeutics 271(2):852-859 (1994)). PS-PLA1 hydrolyzes phosphatidylserine or lysophosphatidyl serine (LysoPS) specifically to produce LysoPS or Glycerol-3-P serine. LysoPS strongly enhances degranulation of rat mast cells induced by concanavalin A and potentiates histamine release (Tamori-Natori et al., J. Biochem (Tokyo) 100(3):581-590 (1986)), and can stimulate sPLA2-elicited histamine release from rat serosal mast cells (Hara et al., Biol. Pharm. Bull. 19(3):474-476 (1996)). LysoPS is an inflammatory lipid mediator (Lloret et al., J. Cell Physiol. 165(1):89-95 (1995)) and sPLA2 has been implicated in inflammation processes (Lloret et al., Toxicon 32(11):1327-1336 (1994)). LysoPI has been shown to stimulate yeast adenylyl cyclase activity with implications for modulating the activity of downstream effector molecules and their interaction with RAS proteins (Resnick and Thomaska, J. Biol. Chem. 269(51):32336-32341 (1994)).
Methods for separating and semi-quantitatively measuring phospholipids such as LysoPA using techniques such as thin-layer chromatography (TLC) followed by gas chromatography (GC) and/or mass spectrometry (MS) are known. For example, lipids may be extracted from the test sample of bodily fluid using extraction procedures such as those described by Bligh and Dyer, Can. J. Biochem. Physiol. 37:911-917 (1959). Thin-layer chromatography may be used to separate various phospholipids, for example as described by Thomas and Holub, Biochim. Biophys. Acta, 1081:92-98 (1991). Phospholipids and lysophospholipids are then visualized on plates, for example using ultraviolet light as described by Gaudette et al., J. Biol. Chem. 268:13773-13776 (1993). Alternatively, lysophospholipid concentrations can be identified by NMR or HPLC following isolation from phospholipids or as part of the phospholipid (Creer and Gross, Lipids 20(12):922-928 (1985) and Bowes et al., J. Biol. Chem. 268(19)13885-13892 (1993)). LysoPA levels have also been determined in ascites from ovarian cancer patients using an assay that relies on LysoPA-specific effects on eukaryotic cells in culture (Mills et al., Cancer Res. 48:1066-1071 (1988)). However, these prior procedures are time-consuming, expensive and variable and typically only semi-quantitative.
Development of a rapid and sensitive assay for lysophospholipid species would facilitate use of these lysophospholipids as markers for cellular activities such as platelet activation and for conditions associated with altered levels of lysophospholipid species. Moreover, such assays would provide a method for determining correlations between altered levels of a lysophospholipid and conditions associated with such altered levels.